scholarly journals The structural dynamics of macropinosome formation and PI3-kinase-mediated sealing revealed by lattice light sheet microscopy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shayne E. Quinn ◽  
Lu Huang ◽  
Jason G. Kerkvliet ◽  
Joel A. Swanson ◽  
Steve Smith ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Three Dimensional ◽  
Light Sheet ◽  
Dimensional Structure ◽  
Minor Role ◽  
Membrane Ruffling ◽  
Light Sheet Microscopy ◽  
Pi3k Activity ◽  
Intracellular Organelles ◽  
A Minor

AbstractMacropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3′-phosphoinositides within ruffles plays a minor role in regulating their morphology. However, 3′-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together, these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.

scholarly journals The structural dynamics of macropinosome formation and PI3-kinase-mediated sealing revealed by lattice light sheet microscopy

2020 ◽  
Author(s):  
Shayne E. Quinn ◽  
Lu Huang ◽  
Jason G. Kerkvliet ◽  
Joel A. Swanson ◽  
Steve Smith ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Three Dimensional ◽  
Light Sheet ◽  
Dimensional Structure ◽  
Membrane Ruffling ◽  
Light Sheet Microscopy ◽  
Pi3k Activity ◽  
Intracellular Organelles ◽  
Early Production ◽  
A Minor

AbstractMacropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through non-specific collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3’-phosphoinositides within ruffles plays a minor in regulating their morphology. However, 3’-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.

scholarly journals The structural dynamics of macropinosome formation and PI3-kinase-mediated sealing revealed by lattice lightsheet microscopy

2020 ◽  
Author(s):  
Shayne Quinn ◽  
Lu Huang ◽  
Jason Kerkvliet ◽  
Joel Swanson ◽  
Steve Smith ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Phosphatidylinositol 3 Kinase ◽  
Membrane Ruffling ◽  
Pi3k Activity ◽  
Intracellular Organelles ◽  
Early Production ◽  
A Minor ◽  
Pharmacologic Inhibition

Abstract Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through non-specific collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3'-phosphoinositides within ruffles plays a minor in regulating their morphology. However, 3'-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.

scholarly journals Multi-color 4D superresolution light-sheet microscopy reveals organelle interactions at isotropic 100-nm resolution and sub-second timescales

2021 ◽  
Author(s):  
Peng Fei
Keyword(s):  
Dimensional Space ◽  
Three Dimensional ◽  
Light Sheet ◽  
Three Dimensions ◽  
Live Cells ◽  
Spatiotemporal Resolution ◽  
Volumetric Imaging ◽  
Light Sheet Microscopy ◽  
Intracellular Organelles ◽  
Microscopy Techniques

Long-term visualization of the dynamic organelle-organelle or protein-organelle interactions throughout the three-dimensional space of whole live cells is essential to better understand their functions, but this task remains challenging due to the limitations of existing three-dimensional fluorescence microscopy techniques, such as an insufficient axial resolution, low volumetric imaging rate, and photobleaching. Here, we present the combination of a progressive deep-learning superresolution strategy with a dual-ring-modulated SPIM design capable of visualizing the dynamics of intracellular organelles in live cells for hours at an isotropic spatial resolution of ~100 nm in three dimensions and a temporal resolution up to ~17 Hz. With a compelling spatiotemporal resolution, we substantially reveal the complex spatial relationships and interactions between the endoplasmic reticulum (ER) and mitochondria throughout live cells, providing new insights into ER-mediated mitochondrial division. We also localized the motion of Drp1 oligomers in three dimensions and observed Drp1-mediated mitochondrial branching for the first time.

scholarly journals Three-dimensional quantification of twisting in the Arabidopsis petiole

2021 ◽  
Author(s):  
Yuta Otsuka ◽  
Hirokazu Tsukaya
Keyword(s):  
Cross Sections ◽  
Three Dimensional ◽  
Light Sheet ◽  
Underlying Mechanism ◽  
Two Dimensions ◽  
Observation Angle ◽  
Differential Growth ◽  
Light Sheet Microscopy ◽  
Definition Of ◽  
Twisting Angle

AbstractOrganisms have a variety of three-dimensional (3D) structures that change over time. These changes include twisting, which is 3D deformation that cannot happen in two dimensions. Twisting is linked to important adaptive functions of organs, such as adjusting the orientation of leaves and flowers in plants to align with environmental stimuli (e.g. light, gravity). Despite its importance, the underlying mechanism for twisting remains to be determined, partly because there is no rigorous method for quantifying the twisting of plant organs. Conventional studies have relied on approximate measurements of the twisting angle in 2D, with arbitrary choices of observation angle. Here, we present the first rigorous quantification of the 3D twisting angles of Arabidopsis petioles based on light sheet microscopy. Mathematical separation of bending and twisting with strict definition of petiole cross-sections were implemented; differences in the spatial distribution of bending and twisting were detected via the quantification of angles along the petiole. Based on the measured values, we discuss that minute degrees of differential growth can result in pronounced twisting in petioles.

scholarly journals Three-Dimensional Cross-Sectional Light-Sheet Microscopy Imaging of the Inflamed Mouse Gut

2017 ◽  
Vol 153 (4) ◽  
pp. 898-900 ◽  
Author(s):  
Sebastian Zundler ◽  
Anika Klingberg ◽  
Daniela Schillinger ◽  
Sarah Fischer ◽  
Clemens Neufert ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Light Sheet ◽  
Cross Sectional ◽  
Microscopy Imaging ◽  
Light Sheet Microscopy

scholarly journals Three Dimensional Fluorescence Imaging Using Multiple Light-Sheet Microscopy

PLoS ONE ◽  
2014 ◽  
Vol 9 (6) ◽  
pp. e96551 ◽  
Author(s):  
Kavya Mohan ◽  
Subhajit B. Purnapatra ◽  
Partha Pratim Mondal
Keyword(s):  
Fluorescence Imaging ◽  
Three Dimensional ◽  
Light Sheet ◽  
Light Sheet Microscopy

scholarly journals Actin-based protrusions of migrating neutrophils are intrinsically lamellar and facilitate direction changes

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Lillian K Fritz-Laylin ◽  
Megan Riel-Mehan ◽  
Bi-Chang Chen ◽  
Samuel J Lord ◽  
Thomas D Goddard ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Light Sheet ◽  
Time Lapse ◽  
Three Dimensions ◽  
Cortical Actin ◽  
Linear Arrangement ◽  
Collagen Matrices ◽  
Flat Surfaces ◽  
Actin Networks ◽  
Light Sheet Microscopy

Leukocytes and other amoeboid cells change shape as they move, forming highly dynamic, actin-filled pseudopods. Although we understand much about the architecture and dynamics of thin lamellipodia made by slow-moving cells on flat surfaces, conventional light microscopy lacks the spatial and temporal resolution required to track complex pseudopods of cells moving in three dimensions. We therefore employed lattice light sheet microscopy to perform three-dimensional, time-lapse imaging of neutrophil-like HL-60 cells crawling through collagen matrices. To analyze three-dimensional pseudopods we: (i) developed fluorescent probe combinations that distinguish cortical actin from dynamic, pseudopod-forming actin networks, and (ii) adapted molecular visualization tools from structural biology to render and analyze complex cell surfaces. Surprisingly, three-dimensional pseudopods turn out to be composed of thin (<0.75 µm), flat sheets that sometimes interleave to form rosettes. Their laminar nature is not templated by an external surface, but likely reflects a linear arrangement of regulatory molecules. Although we find that Arp2/3-dependent pseudopods are dispensable for three-dimensional locomotion, their elimination dramatically decreases the frequency of cell turning, and pseudopod dynamics increase when cells change direction, highlighting the important role pseudopods play in pathfinding.

Intracellular calcium in cultured rabbit oviduct epithelial cells

1996 ◽  
Vol 8 (2) ◽  
pp. 243 ◽  
Author(s):  
CJ Dickens ◽  
CI Cox ◽  
HJ Leese
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Intracellular Calcium ◽  
Fluid Secretion ◽  
Minor Role ◽  
Ion Substitution ◽  
Secretory Type ◽  
Oviduct Epithelial Cells ◽  
A Minor ◽  
Oviduct Fluid

Oviduct fluid is the medium in which fertilization and early embryonic development occur but little is known about the ionic basis of fluid secretion or its control. Since calcium ions (Ca2+) are involved in the mechanism of secretion in other epithelia, the intracellular calcium concentration ([Ca2+]i) was measured in single, rabbit oviduct epithelial cells in primary culture using the fluorescent dye Fura-2. The resting [Ca2+]i was constant (115 nM) in cells cultured for 2-7 days. Ion substitution experiments demonstrated the presence of a Na+/Ca(2+)-exchange system in the plasma membrane, whereas influx through channels was found to have only a minor role maintaining the resting [Ca2+]i. The addition of dibutyryl cAMP (db cAMP) induced two types of response: the first was an increase in [Ca2+]i, dependent on the presence of extracellular Ca2+, and the second was a zero response. Extracellular ATP induced a transient increase in [Ca2+]i owing to the release of Ca2+ from intracellular stores and Ca2+ entering the cell across the plasma membrane. It is proposed that these effects may be due to the presence of two types of cell in culture-the ciliated and non-ciliated (secretory type) oviduct epithelial cells.

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